Somatic Cell and Molecular Genetics

, Volume 21, Issue 5, pp 299–307 | Cite as

Efficient modification of the APRT gene by FLP/FRT site-specific targeting

  • Raymond V. Merrihew
  • R. Geoffrey Sargent
  • John H. Wilson


The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5×10−5, which were 6–22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.


Gene Target Southern Blotting Homologous Recombination Separate Experiment Additional Marker 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Adair, G., Nairn, R., Wilson, J., Seidman, M., Brotherman, K., MacKinnon, C., and Scheerer, J. (1989).Proc. Natl. Acad. Sci. USA 86:4574–4578.PubMedGoogle Scholar
  2. 2.
    Adair, G., Nairn, R., Wilson, J., Scheerer, J., and Brotherman, K. (1990).Somat. Cell Mol. Genet. 16:437–441.CrossRefPubMedGoogle Scholar
  3. 3.
    Pennington, S. and Wilson, J. (1991).Proc. Natl. Acad. Sci. USA 88:9498–9502.PubMedGoogle Scholar
  4. 4.
    Sauer, B., and Henderson, N. (1990).New Biol. 2:441–449.PubMedGoogle Scholar
  5. 5.
    O'Gorman, S., Fox, D., and Wahl, G. (1991).Science 251:1351–1355.PubMedGoogle Scholar
  6. 6.
    Chambers, C. (1994).BioEssays 16:865–868.CrossRefPubMedGoogle Scholar
  7. 7.
    Fukushige, S., and Sauer, B. (1992).Proc. Natl. Acad. Sci. USA 89:7905–7909.PubMedGoogle Scholar
  8. 8.
    Ludwig, D., and Stringer, J. (1994).Somat. Cell Mol. Genet. 20:11–25.CrossRefPubMedGoogle Scholar
  9. 9.
    Smith, A., DeSousa, M., Kwabi-Addo, B., Happell-Parton, A., Impey, H., and Rabbitts, P. (1995).Nature Genet. 9:376–385.CrossRefPubMedGoogle Scholar
  10. 10.
    Fiering, S., Kim, C., Epner, E., and Groudins, M. (1993).Proc. Natl. Acad. Sci. USA 90:8469–8473.PubMedGoogle Scholar
  11. 11.
    Gu, H., Zou, Y.-R., and Rajewsky, K. (1993).Cell 73:1155–1164.CrossRefPubMedGoogle Scholar
  12. 12.
    Lakso, M., Sauer, B., Monsinger Jr., B., Lee, E., Manning, R., Yu, S.-H., Mulder, K., and Westphal, H. (1992).Proc. Natl. Acad. Sci. USA 89:6232–6236.PubMedGoogle Scholar
  13. 13.
    Orban, P., Chui, D., and Marth, J. (1992).Proc. Natl. Acad. Sci. USA 89:6861–6865.PubMedGoogle Scholar
  14. 14.
    Gu, H., Marth, J., Orban, P., Mossmann, H., and Rajewsky, K. (1994).Science 265:103–106.PubMedGoogle Scholar
  15. 15.
    Chu, G., Hayakawa, H., and Berg, P. (1987).Nucleic Acids Res. 15:1311–1326.PubMedGoogle Scholar
  16. 16.
    Sambrook, J., Fritsch, E., and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).Google Scholar
  17. 17.
    Merrihew, R., Marburger, K., Pennington, S., Roth D., and Wilson, J.Mol. Cell. Biol., in press.Google Scholar
  18. 18.
    Baubonis, W., and Sauer, B. (1993).Nucleic Acids Res. 21:2025–2029.PubMedGoogle Scholar
  19. 19.
    Godwin, A., Bollag, R., Christie, D.-M., and Liskay, R. (1994).Proc. Natl. Acad. Sci. USA 91:12554–12558.PubMedGoogle Scholar
  20. 20.
    Subramani, S., and Rubnitz, J. (1985).Mol. Cell. Biol. 5:659–666.PubMedGoogle Scholar
  21. 21.
    Lacy, E., Roberts, S., Evans, E., Burtenshaw, M., and Constantini, F. (1983).Cell 34:343–358.CrossRefPubMedGoogle Scholar
  22. 22.
    Schlake, T., and Bode, J. (1994).Biochemistry 33:12746–12751.CrossRefPubMedGoogle Scholar
  23. 23.
    Sauer, B., and Henderson, N. (1989).Nucleic Acids Res. 17:147–161.PubMedGoogle Scholar
  24. 24.
    Stark, W., Boocock, M., and Sherratt, D. (1992).Trends Genet. 8:432–439.CrossRefPubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Raymond V. Merrihew
    • 1
  • R. Geoffrey Sargent
    • 1
  • John H. Wilson
    • 1
  1. 1.The Verna and Marrs McLean Department of BiochemistryBaylor College of MedicineHouston

Personalised recommendations